| Summary: | This study aimed to implement a multimodal <sup>1</sup>H/HP-<sup>13</sup>C imaging protocol to augment the serial monitoring of patients with glioma, while simultaneously pursuing methods for improving the robustness of HP-<sup>13</sup>C metabolic data. A total of 100 <sup>1</sup>H/HP [1-<sup>13</sup>C]-pyruvate MR examinations (104 HP-<sup>13</sup>C datasets) were acquired from 42 patients according to the comprehensive multimodal glioma imaging protocol. Serial data coverage, accuracy of frequency reference, and acquisition delay were evaluated using a mixed-effects model to account for multiple exams per patient. Serial atlas-based HP-<sup>13</sup>C MRI demonstrated consistency in volumetric coverage measured by inter-exam dice coefficients (0.977 ± 0.008, mean ± SD; four patients/11 exams). The atlas-derived prescription provided significantly improved data quality compared to manually prescribed acquisitions (<i>n</i> = 26/78; <i>p</i> = 0.04). The water-based method for referencing [1-<sup>13</sup>C]-pyruvate center frequency significantly reduced off-resonance excitation relative to the coil-embedded [<sup>13</sup>C]-urea phantom (4.1 ± 3.7 Hz vs. 9.9 ± 10.7 Hz; <i>p</i> = 0.0007). Significantly improved capture of tracer inflow was achieved with the 2-s versus 5-s HP-<sup>13</sup>C MRI acquisition delay (<i>p</i> = 0.007). This study demonstrated the implementation of a comprehensive multimodal <sup>1</sup>H/HP-<sup>13</sup>C MR protocol emphasizing the monitoring of steady-state/dynamic metabolism in patients with glioma.
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